microorganisms Article Land-Use Type Drives Soil Population Structures of the Entomopathogenic Fungal Genus Metarhizium María Fernández-Bravo 1 , Florian Gschwend 1 , Johanna Mayerhofer 1 , Anna Hug 2, Franco Widmer 1 and Jürg Enkerli 1,* 1 Molecular Ecology, Agroscope, CH-8046 Zürich, Switzerland; [email protected] (M.F.-B.); fl[email protected] (F.G.); [email protected] (J.M.); [email protected] (F.W.) 2 Swiss Soil Monitoring Network (NABO), Agroscope, CH-8046 Zürich, Switzerland; [email protected] * Correspondence: [email protected] Abstract: Species of the fungal genus Metarhizium are globally distributed pathogens of arthropods, and a number of biological control products based on these fungi have been commercialized to control a variety of pest arthropods. In this study, we investigate the abundance and population structure of Metarhizium spp. in three land-use types—arable land, grassland, and forest—to provide detailed information on habitat selection and the factors that drive the occurrence and abundance of Metarhizium spp. in soil. At 10 sites of each land-use type, which are all part of the Swiss national soil-monitoring network (NABO), Metarhizium spp. were present at 8, 10, and 4 sites, respectively. On average, Metarhizium spp. were most abundant in grassland, followed by forest and then arable land; 349 Metarhizium isolates were collected from the 30 sites, and sequence analyses of the Citation: Fernández-Bravo, M.; nuclear translation elongation factor 1α gene, as well as microsatellite-based genotyping, revealed Gschwend, F.; Mayerhofer, J.; Hug, A.; the presence of 13 Metarhizium brunneum, 6 Metarhizium robertsii, and 3 Metarhizium guizhouense Widmer, F.; Enkerli, J. Land-Use Type Drives Soil Population Structures of multilocus genotypes (MLGs). With 259 isolates, M. brunneum was the most abundant species, and the Entomopathogenic Fungal Genus significant differences were detected in population structures between forested and unforested sites. Metarhizium. Microorganisms 2021, 9, Among 15 environmental factors assessed, C:N ratio, basal respiration, total carbon, organic carbon, 1380. https://doi.org/10.3390/ and bulk density significantly explained the variation among the M. brunneum populations. The microorganisms9071380 information gained in this study will support the selection of best-adapted isolates as biological control agents and will provide additional criteria for the adaptation or development of new pest Academic Editor: Victor Glupov control strategies. Received: 28 May 2021 Keywords: M. brunneum; M. robertsii; M. guizhouense; microsatellite; SSR; EF-1alpha; abiotic factors; Accepted: 22 June 2021 arable land; grassland; forest; biological control Published: 25 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. The genus Metarhizium Sorok¯ın (Hypocreales: Clavicipitaceae) includes more than 30 described species (including both asexual and sexual states) that are pathogenic to arthropods, particularly insects and some arachnids [1,2]. The wide host spectrum of Metarhizium spp. includes many important crop pests such as Helicoverpa armigera (Hübner, 1805), Diabrotica virgifera virgifera (LeConte, 1868), Agriotes spp. (Eschscholtz, 1829), and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. also termites, cockroaches, and even disease-transmitting insects such as tse-tse flies [2–7]. This article is an open access article Based on the pathogenicity of this fungus, a number of biological control agents have been distributed under the terms and developed as commercial mycoinsecticides and mycoacaricides to control pest arthropods conditions of the Creative Commons worldwide (see [8] and https://www.eppo.int; https://www.epa.gov). Attribution (CC BY) license (https:// Soil is considered the main habitat and reservoir of Metarhizium spp., and they have creativecommons.org/licenses/by/ been isolated from very different ecosystems, from tropical to temperate, semiarid, and 4.0/). artic areas [5,9]. Besides being entomopathogenic, Metarhizium spp. exhibit a pronounced Microorganisms 2021, 9, 1380. https://doi.org/10.3390/microorganisms9071380 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1380 2 of 19 saprophytic lifestyle, and, for some isolates and/or species, symbiotic interactions with plants as rhizosphere colonizers and/or endophytes have been reported [9,10]. Studies have demonstrated, for instance, that isolates of M. robertsii J.F. Bisch., S.A. Rehner & Humber, M. brunneum Petch, and M. guizhouense Q.T. Chen & H.L. Guo can provide insect- derived nitrogen to plants [11,12], increase plant growth and productivity [13,14], as well as protect plants from environmental stresses, for example, in saline environments [15,16]. The versatile roles Metarhizium spp. may have emphasize the beneficial potential and value of its presence, particularly in agricultural habitats [17]. Arable land, grassland, and forests represent major anthropogenic land-use types (LUTs) and cover about 40% of the global land area [18]. A number of studies have re- ported possible effects of LUTs on the presence and abundance of Metarhizium spp. Factores responsible includ crop types, farming practices management intensity, tillage, and the application of pesticides or fertilizers [19–22]. For instance, it has been consistently reported that Metarhizium spp. occur at higher abundances in unforested, sun-exposed LUTs or open land areas such as arable- and grasslands compared to forests, where their abundances are lower or sometimes not even detected [17,19,23–26]. The abundance of Metarhizium spp. has been more prominent in unforested semi-natural habitats such as permanent grassland and field margins compared to arable land in Switzerland [27,28]. Furthermore, there is evidence from field studies in Norway indicating a higher abundance of entomopathogenic fungi such as M. anisopliae, Beauveria bassiana (Bals.-Criv.) Vuill., or Tolypocladium cylindrospo- rum W. Gams in soils of organically managed fields compared to conventionally managed fields [29]. Several studies have suggested that the application of organic fertilizers may be the main driving factor for the increased abundance of Metarhizium spp. [22,30]. While the overall abundance of Metarhizium spp. in different LUTs has been explored in various studies, information on the species composition of Metarhizium communities and within-species diversity in different LUTs is limited. In addition, many of these studies have been performed prior to the establishment of the current Metarhizium species concept, which has precluded a comparison of data from these different periods. Nevertheless, the results reported by Cabrera-Mora et al. [31] have indicated that cropping systems can affect species abundances, as M. brunneum was isolated predominantly from soil where beans were grown and M. robertsii from soils where maize was grown. Similarly, Wyre- bek et al. [32] reported that M. robertsii, M. brunneum, and M. guizhouense were closely associated with soils from grassland, shrubs, or trees, respectively. In a study performed in Ontario, Canada, two genetically distinct groups were found to be associated with agricultural unforested field habitats and forested soils, respectively [23,33,34]. Isolates of the two genetic groups were subsequently described as M. brunneum and M. robertsii [35]. Studies assessing within-species diversity have mostly focused on individual fields or agricultural systems only, e.g., an agricultural field in Denmark [36] and a strawberry field in Brazil [37]. A comparison of the within-species diversity of M. brunneum and M. robertii between agricultural and adjacent natural habitats has been performed in different ecologi- cal regions in northwestern North America [38]. While considerable genotype diversity has been reported for both species in both habitats, no significant differences have been detected between them. Information on the number of species, their population structures, and distributions in different LUTs remains limited. This information, however, is impor- tant to understand the interaction of ecological conditions and Metarhizium population structures. In turn, it may allow the improvement of existing habitats or the development of new habitats and/or LUT-adapted biological control approaches, including conservation biological control [17]. Conditions in a particular soil or at a particular site are affected, on the one hand, by anthropogenic activities and, on the other hand, by different environmental factors, i.e., abiotic (physical and chemical), biotic (derived from organisms), as well as climatic (humidity, temperature, and solar radiation) factors. All these factors may have direct effects on the occurrence, abundance, propagation, persistence, and even virulence of Metarhizium populations [24,39–42]. Soil physical, chemical, and biological factors, such Microorganisms 2021, 9, 1380 3 of 19 as soil texture, pH, and organic matter content, belong to the most intensively studied factors in this context. However, various reports have not been able to provide consistent information, and, in many cases, they have been contradictory. For example, organic matter, which represents an important source of organic carbon in soil, has been reported to negatively correlate with the abundance of Metarhizium